Hypercholesterolemia increases mitochondrial oxidative stress and enhances the MPT response in the porcine myocardium: beneficial effects of chronic exercise

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We tested the hypothesis that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response and that exercise training could reverse this phenotype. MPT was assessed by mitochondrial swelling in response to 10-100 μM Ca(2+). FH pigs did show an increased MPT response to Ca(2+) that was associated with decreases in the expression of the putative MPT pore components mitochondrial phosphate carrier (PiC) and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation, as well as decreased levels of reduced GSH in cardiac mitochondria. Expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin-2 (Trx2), and peroxiredoxin-3 (Prx3) was greatly reduced in the FH pigs. In contrast, cytosolic catalase expression and activity were increased. However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce mitochondrial oxidative stress, and return MnSOD, Trx2, Prx3, and catalase expression/activities to normal. We conclude that FH reduces mitochondrial antioxidants, increases mitochondrial oxidative stress, and enhances the MPT response in the porcine myocardium, and that exercise training can reverse these detrimental alterations.     

In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase

We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate. The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase.     

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% β-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.     

Methenyl-tetrahydromethanopterin cyclohydrolase in cell extracts of Methanobacterium

Cell extracts of Methanobacterium thermoautotrophicum possess a methenyl-tetrahydromethanopterin (methenyl-H4MPT) cyclohydrolase. The enzyme catalyzes the hydrolysis of methenyl-H4MPT to formyltetrahydromethanopterin (formyl-H4MPT). The reaction is reversible and both the rate and extent of the reaction depend on the pH and the buffer used. Similarly, the nonenzymatic hydrolysis of methenyl-H4MPT is highly dependent on pH and buffer. An active derivative of methenyl-H4MPT was obtained in 94% yield by reacting H4MPT with formic acid in the presence of excess acetic acid under anoxic conditions at 80 degrees C for 3 h. H NMR spectroscopy and fast atom bombardment mass spectrometry revealed the product to be a derivative of methenyl-H4MPT which had lost the alpha-hydroxyglutarylphosphate unit. In spite of this loss, this derivative served both as a substrate for methanogenesis and for the cyclohydrolase. Comparison of the properties of the products of the enzymatic and nonenzymatic hydrolyses indicates that the enzymatic reaction yields N5-formyl-H4MPT whereas the nonenzymatic reaction yields N10-formyl-H4MPT.     

Limited mitochondrial permeabilization is an early manifestation of palmitate-induced lipotoxicity in pancreatic beta-cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.     

Crystal structure of a major secreted protein of Mycobacterium tuberculosis-MPT63 at 1.5-A resolution

MPT63 is a small, major secreted protein of unknown function from Mycobacterium tuberculosis that has been shown to have immunogenic properties and has been implicated in virulence. A BLAST search identified that MPT63 has homologs only in other mycobacteria, and is therefore mycobacteria specific. As MPT63 is a secreted protein, mycobacteria specific, and implicated in virulence, MPT63 is an attractive drug target against the deadliest infectious disease, tuberculosis (TB). As part of the TB Structural Genomics Consortium, the X-ray crystal structure of MPT63 was determined to 1.5-Angstrom resolution with the hope of yielding functional information about MPT63. The structure of MPT63 is an antiparallel beta-sandwich immunoglobulin-like fold, with the unusual feature of the first beta-strand of the protein forming a parallel addition to the small antiparallel beta-sheet. MPT63 has weak structural similarity to many proteins with immunoglobulin folds, in particular, Homo sapiens beta2-adaptin, bovine arrestin, and Yersinia pseudotuberculosis invasin. Although the structure of MPT63 gives no conclusive evidence to its function, structural similarity suggests that MPT63 could be involved in cell-host interactions to facilitate endocytosis/phagocytosis.     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Synthesis of adenylated molybdopterin: an essential step for molybdenum insertion

The molybdenum cofactor (Moco) is part of the active site of all molybdenum (Mo)-dependent enzymes, except nitrogenase. Moco consists of molybdopterin (MPT), a phosphorylated pyranopterin with an enedithiolate coordinating Mo and it is synthesized by an evolutionary old multistep pathway. The plant protein Cnx1 from Arabidopsis thaliana catalyzes with its two domains (E and G) the terminal step of Moco biosynthesis, the insertion of Mo into MPT. Recently, the high-resolution MPT-bound structure of the Cnx1 G domain (Cnx1G) has been determined (Kuper, J., Llamas, A., Hecht, H. J., Mendel, R. R., and Schwarz, G. (2004) Nature 430, 803-806). Besides defining the MPT-binding site a novel and unexpected modification of MPT has been identified, adenylated MPT. Here we demonstrate that it is Cnx1G that catalyzes the adenylation of MPT. In vitro synthesized MPT was quantitatively transferred from Escherichia coli MPT synthase to Cnx1G. The subsequent adenylation reaction by Cnx1G was Mg(2+)- and ATP-dependent. Whereas Mn(2+) could partially replace Mg(2+), ATP was the only nucleotide accepted by Cnx1G. Consequently the formation of pyrophosphate was demonstrated, which was dependent on the ability of Cnx1G to bind MPT. Pyrophosphate, either formed in the reaction or added externally, inhibited the Cnx1G-catalyzed MPT adenylation reaction. Catalytically inactive Cnx1G mutant variants showed impaired MPT adenylation confirming that MPT-AMP is the reaction product of Cnx1G. Therefore Cnx1G is a MPT adenylyltransferase catalyzing the activation of MPT, a universal reaction in the Moco synthetic pathway because Cnx1G is able to reconstitute also bacterial and mammalian Moco biosynthesis.     

Ammonia Neurotoxicity and the Mitochondrial Permeability Transition

Ammonia is a neurotoxin that predominantly affects astrocytes. Disturbed mitochondrial function and oxidative stress, factors implicated in the induction of the mitochondrial permeability transition (MPT), appear to be involved in the mechanism of ammonia neurotoxicity. We have recently shown that ammonia induces the MPT in cultured astrocytes. To elucidate the mechanisms of the MPT, we examined the role of oxidative stress and glutamine, a byproduct of ammonia metabolism. The ammonia-induced MPT was blocked by antioxidants, suggesting a causal role of oxidative stress. Direct application of glutamine (4.5-7.0 mM) to cultured astrocytes increased free radical production and induced the MPT. Treatment of astrocytes with the mitochondrial glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, completely blocked free radical formation and the MPT, suggesting that high ammonia concentrations in mitochondria resulting from glutamine hydrolysis may be responsible for the effects of glutamine. These studies suggest that oxidative stress and glutamine play major roles in the induction of the MPT associated with ammonia neurotoxicity.     

How an enzyme binds the C1 carrier tetrahydromethanopterin. Structure of the tetrahydromethanopterin-dependent formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.     

A bioenergetic model of the mitochondrial population undergoing permeability transition

Mitochondrial permeability transition (MPT) is a highly regulated complex phenomenon that is a type of ischemia/reperfusion injury that can lead to cell death and ultimately organ dysfunction. A novel population transition and detailed permeability transition pore regulation model were integrated with an existing bioenergetics model to describe MPT induction under a variety of conditions. The framework of the MPT induction model includes the potential states of the mitochondria (aggregated, orthodox and post-transition), their transitions from one state to another as well as their interaction with the extra-mitochondrial environment. The model encodes the three basic necessary conditions for MPT: a high calcium load, alkaline matrix pH and circumstances which favor de-energization. The MPT induction model was able to reproduce the expected bioenergetic trends observed in a population of mitochondria subjected to conditions that favor MPT. The model was corroborated and used to predict that MPT in an acidic environment is mitigated by an increase in activity of the mitochondrial potassium/hydrogen exchanger. The model was also used to present the beneficial impact of reducing the duration mitochondria spend in the orthodox state on preserving the extra-mitochondrial ATP levels. The model serves as a tool for investigators to use to understand the MPT induction phenomenon, explore alternative hypotheses for PTP regulation, as well as identify endogenous pharmacological targets and evaluate potential therapeutics for MPT mitigation.     

A sulfurtransferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z in Escherichia coli

It has been shown that conversion of precursor Z to molybdopterin (MPT) by Escherichia coli MPT synthase entails the transfer of the sulfur atom of the C-terminal thiocarboxylate from the small subunit of the synthase to generate the dithiolene group of MPT and that the moeB mutant of E. coli contains inactive MPT synthase devoid of the thiocarboxylate. The data presented here demonstrate that l-cysteine can serve as the source of the sulfur for the biosynthesis of MPT in vitro but only in the presence of a persulfide-containing sulfurtransferase such as IscS, cysteine sulfinate desulfinase (CSD), or CsdB. A fully defined in vitro system has been developed in which an inactive form of MPT synthase can be activated by incubation with MoeB, Mg-ATP, l-cysteine, and one of the NifS-like sulfurtransferases, and the addition of precursor Z to the in vitro system gives rise to MPT formation. The use of radiolabeled l-[(35)S]cysteine has demonstrated that both sulfurs of the dithiolene group of MPT originate from l-cysteine. It was found that MPT can be produced from precursor Z in an E. coli iscS mutant strain, indicating that IscS is not required for the in vivo sulfuration of MPT synthase. A comparison of the ability of the three sulfurtransferases to provide the sulfur for MPT formation showed the highest activity for CSD in the in vitro system.     

The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified to electrophoretic homogeneity from cells of Methanobacterium thermoautotrophicum. The enzyme is a tetramer of similar or identical subunits (Mr = 41,000). The equilibrium favors transfer of the formyl group to tetrahydromethanopterin (H4MPT) at physiological pH. The product of formyl transfer by the purified enzyme was shown by a number of criteria to be 5-formyl-H4MPT, as opposed to 10-formyl-H4MPT or 5,10-methenyl-H4MPT. Reconstitution of a portion of the methanogenic C1 cycle was effected by combining purified formyltransferase, methenyl-H4MPT cyclohydrolase, formylmethanofuran, and H4MPT to give methenyl-H4MPT. Additional reconstitution experiments established that the formyltransferase is an essential enzyme for the conversion of carbon dioxide to methane. In conjunction with previously published data (Donnelly, M.I., Escalante-Semerena, J.C., Rinehart, K. L., Jr., and Wolfe, R.S. (1985) Arch. Biochem. Biophys. 242, 430-439), these data substantiate the role of 5-formyl-H4MPT as an intermediate of methanogenesis.     

The role of the mitochondrial permeability transition in cell death

The mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that occurs in response to increased calcium load and redox stress. Currently, two models of the MPT exist including the, largely hypothetical, native proteinaceous pore model and the oxidized inner membrane protein model which may reflect the extremes in a continuum of changes that occur to the inner membrane prior to its permeabilization. Here I discuss evidence that the MPT per se leads to necrosis, but not cytochrome c release and apoptosis. However, data also suggest that signaling crosstalk between the MPT and Bcl-2 family proteins occurs indicating an important role for the MPT in apoptosis.     

Anticancer Activity of MPT0E028, a Novel Potent Histone Deacetylase Inhibitor, in Human Colorectal Cancer HCT116 Cells In Vitro and In Vivo

Recently, histone deacetylase (HDAC) inhibitors have emerged as a promising class of drugs for treatment of cancers, especially subcutaneous T-cell lymphoma. In this study, we demonstrated that MPT0E028, a novel N-hydroxyacrylamide-derived HDAC inhibitor, inhibited human colorectal cancer HCT116 cell growth in vitro and in vivo. The results of NCI-60 screening showed that MPT0E028 inhibited proliferation in both solid and hematological tumor cell lines at micromolar concentrations, and was especially potent in HCT116 cells. MPT0E028 had a stronger apoptotic activity and inhibited HDACs activity more potently than SAHA, the first therapeutic HDAC inhibitor proved by FDA. In vivo murine model, the growth of HCT116 tumor xenograft was delayed and inhibited after treatment with MPT0E028 in a dose-dependent manner. Based on in vivo study, MPT0E028 showed stronger anti-cancer efficacy than SAHA. No significant body weight difference or other adverse effects were observed in both MPT0E028-and SAHA-treated groups. Taken together, our results demonstrate that MPT0E028 has several properties and is potential as a promising anti-cancer therapeutic drug.     

Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins.

Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase. In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The recently solved crystal structure of MPT synthase (Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H. (2000) Nat. Struct. Biol. 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE). According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z. Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD. Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE. Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT. Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography. A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase.     

Cytochrome bc(1) regulates the mitochondrial permeability transition by two distinct pathways

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc(1) regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc(1). The ROS producing mechanism in cytochrome bc(1) involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc(1) by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc(1). These results indicate that electron flux through cytochrome bc(1) regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.     

Thermodynamic analysis of subunit interactions in Escherichia coli molybdopterin synthase

The molybdopterin (MPT) synthase complex in Escherichia coli consists of two MoaE subunits and two MoaD subunits in a heterotetrameric structure with the two MoaE subunits forming a central dimer. Each MoaD subunit binds to a single MoaE molecule to form two identical MoaE/MoaD interfaces. Here we define the thermodynamic properties of the interaction between MoaE and MoaD in MPT synthase using a H/D exchange and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy based method termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX-derived protein folding free energies and m values are reported for MoaE in the presence and absence of MoaD and MoaD-SH, the thiocarboxylated form of MoaD that is essential for the catalytic activity of MPT synthase. The protein folding free energy measurements were used to calculate a dissociation constant of 17 +/- 7 microM for the binding of MoaD to MoaE in inactive MPT synthase and a dissociation constant of 2.6 +/- 0.9 microM for the binding of MoaD-SH to MoaE in active MPT synthase. The increased binding affinity of MoaD-SH for MoaE is consistent with a previously proposed mechanism for the MPT synthase reaction. Using the increased m values exhibited by MoaE in the presence of either MoaD subunit, the solvent accessible surface area buried upon formation of the subunit interface in MPT synthase was estimated to be 2378 A(2) for inactive MPT synthase and 4117 A(2) for active MPT synthase.     

Alloxan-induced mitochondrial permeability transition triggered by calcium, thiol oxidation, and matrix ATP

In addition to their critical function in energy metabolism, mitochondria contain a permeability transition pore, which is regulated by adenine nucleotides. We investigated conditions required for ATP to induce a permeability transition in mammalian mitochondria. Mitochondrial swelling associated with mitochondria permeability transition (MPT) was initiated by adding succinate to a rat liver mitochondrial suspension containing alloxan, a diabetogenic agent. If alloxan was added immediately with or 5 min after adding succinate, MPT was strikingly decreased. MPT induced by alloxan was inhibited by EGTA and several agents causing thiol oxidation, suggesting that alloxan leads to permeability transition through a mechanism dependent on Ca(2+) uptake and sulfhydryl oxidation. Antimycin A and cyanide, inhibitors of electron transfer, carbonyl cyanide m-chlorophenylhydrazone, and oligomycin all inhibited MPT. During incubation with succinate, alloxan depleted ATP in mitochondria after an initial transient increase. However, in a mitochondrial suspension containing EGTA, ATP significantly increased in the presence of alloxan to a level greater than that of the control. These results suggest the involvement of energized transport of Ca(2+) in the MPT initiation. Addition of exogenous ATP, however, did not trigger MPT in the presence of alloxan and had no effect on MPT induced by alloxan. We conclude that alloxan-induced MPT requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca(2+).     

The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase

Methods have been devised to examine the spectral properties and state of reduction of the pterin ring of molybdopterin (MPT) in milk xanthine oxidase and the Mo-containing domain of rat liver sulfite oxidase. The absorption spectrum of the native pterin was visualized by difference spectroscopy of each protein, denatured anaerobically in 6 M guanidine hydrochloride (GdnHCl), versus a sample containing the respective apoprotein and other necessary components. The state of reduction of MPT was also probed using 2,6-dichlorobenzenoneindophenol (DCIP) to measure reducing equivalents/MPT, after anaerobic denaturation of the protein in GdnHCl in the presence or absence of Hg2+. In the case of xanthine oxidase the data indicate that the terminal sulfide ligand of Mo causes the reduction of a native dihydro form of MPT to the tetrahydro level. This reduction does not occur if Hg2+ is added prior to denaturation of the protein. Based on its observed behavior, the native MPT in the Mo cofactor of xanthine oxidase is postulated to exist as a quinonoid dihydropterin. Quantitation of DCIP reduction by MPT of Mo fragment of sulfite oxidase showed a two-electron oxidation of MPT, even when the Mo fragment was denatured in the presence of Hg2+ to prevent internal reduction reactions due to sulfhydryls or sulfide. Difference spectra of DCIP-treated versus untreated Mo fragment showed that MPT had been fully oxidized. These data indicate that the native MPT in sulfite oxidase must be a dihydro isomer different from that in xanthine oxidase.